Criteria for early diagnosis of third molar agenesis: a retrospective radiographic study

ABSTRACT Objective: To explore the association between chronological, dental and skeletal ages and early diagnosis of third molars agenesis. Material and Methods: This retrospective radiographic study comprised a sample of 282 Portuguese patients (122 males and 160 females) who sought orthodontic treatment between 2007 and 2018. Each participant had panoramic and lateral cephalometric radiographs performed before and after the age of 14 years. The chronological age was categorized into three intervals between 11.0 and 13.11 years of age. The full eruption of the four-second molars was used as a criterion in determining dental age. Skeletal age was verified by the vertebral maturation method. The diagnosis of agenesis of third molars was initially performed by observation of the initial panoramic radiography undertaken before the age of 14 years. Subsequently, the diagnosis of agenesis of third molars was confirmed by visualizing the second panoramic radiography, obtained after the age of 14 years. The association between the accuracy of the diagnosis and the chronological, dental and skeletal ages was evaluated using the chi-square test, at a 5% significance level. Results: No significant association between chronological age and alterations in the diagnosis of third molar agenesis was identified. However, there was a significant association between third molar agenesis and both dental age (p<0.001) and skeletal age (p=0.006). Conclusion: The eruption of the four-second molars and the peak of growth could be considered as criteria for early diagnosis of third molar agenesis, whereas chronological age is not a reliable diagnostic indicator.


INTRODUCTION
Selective tooth agenesis is one of the most prevalent developmental anomalies in humans. It can arise following disruptions at the early stages of tooth development, i.e. initiation and proliferation. 1 Congenital absence of the third molar is the most prevalent type of hypodontia, with reported rates reaching 50% in some affected groups. 2 In addition, Hellman 3 reported that subjects with congenitally missing third molars are 13 times more prone to have agenesia of other teeth. Third molar germ formation usually starts around 11 years of age in about 90% of individuals, and it commonly emerges in the oral cavity at 18-20 years of age. 4 It is generally accepted that agenesis of permanent teeth is strongly associated with the development of malocclusion. 5 Therefore, clinicians, in particular orthodontists, ought to evaluate the whole dentition, including the existence or absence of a third molar. 6 Moreover, in orthodontics, it has become increasingly recognized that beginning a treatment at the optimal time can be as critical as selecting a specific treatment protocol. 7,8 Biological indicators such as sexual maturation, chronological age, 9 dental development, 10 and skeletal development, 11,12 are some of the criteria most commonly used to identify the maturation stage, thereby contributing to the planning of an orthodontic treatment at the optimal time. 7 Dumas M, Mariano-Pereira P, Bugaighis I, Fernandes-Retto P, Proença L -Criteria for early diagnosis of third molar agenesis: a retrospective radiographic study Many researchers have attempted to identify the earliest age at which a third molar germ can be radiographically visualized. 13 The age of onset of third molar development is very variable, 14 and there is no consensus among authors about the chronological age at which a third molar tooth can be considered congenitally missing. 15,16,17 The most frequently reported critical age is 14 years. 15,16 However, Richardson 17 observed that in the absence of a third molar germ at 10 years of age, the probability of the tooth being congenitally missing is 50%. Bolaños et al. 14 proposed that the radiographic diagnosis of agenesia can be performed at 13 years of age. Nevertheless, a weak correlation between chronological age and development of the third molar has been observed. 14,18 Estimating dental age in children and adolescents is based mainly on dental calcification observed in radiographs and the timing of dental eruption. 19 Generally, panoramic radiographs are used to evaluate the stages of crown and root development. 10 Published studies show that third molar congenital absence varies in different populations 14,20 Several studies have reported a significant association between calcification delay and eruption of first premolar or second molar with third molar agenesis. 15,17,19 Good quality lateral cephalometric radiographs, with a clear view of the cervical spine, have been widely used to evaluate the stage of Cervical Vertebral Maturation (CVM) and to correlate this with the stage of skeletal maturity. The advantage of this approach is that the same cephalometric radiograph is used for both purposes, which obviates the need for a second radiograph, thereby reducing the degree of radiation exposure. A significant amount of studies had verified that the CVM method is a reliable means of assessing circumpubertal growth levels and the phases of skeletal maturity. Baccetti et al. 11 CVM assessment has become the most widely used method for assessing skeletal age. 12,21,22 However, there is a clear lack of studies that explore the role of skeletal age in diagnosing agenesis of the third molars. In this context, the main objective of the present study was to explore the association between chronological, dental and skeletal age and early diagnosis of third molar agenesis.
The following null hypotheses were tested: 1. It is not possible to make a reliable diagnosis of third molar agenesis in patients aged between 11.0 and 13.11 years using chronological age alone.  The sample size for this study was calculated for 80% power and 5% significance level, considering an effect size of 0.2 on the diagnosis of third molar agenesis, as a function of dental age. According to the calculation, the required minimum sample size was 273 subjects (118 males and 155 females), while adjusting for sex. Chronologically, the sample was divided into three groups: Group 1, aged between 11.0 and 11.11 years; Group 2, aged between 12.0 and 12.11; Group 3, between 13.0 and 13.11 years. Regarding dental age, the cohort was categorized into two working groups: Group I, without full eruption of the four second molars; Group II, individuals with eruption of the four second molars. Full eruption of the four second molars was considered to have occurred when these teeth reached the occlusal plane, i.e., when radiographically the cusps of the second molars contacted the plane passing through the cusp of the first premolar and the cusps of the first molar. 10 The stages of vertebral maturation (skeletal age) were visualized using two-dimensional lateral cephalometric radiographs and categorized according to Baccetti et al. 11,12 for the second (C2), third (C3), and fourth (C4) cervical vertebrae. Six maturational stages of these three cervical vertebrae were identified based on the morphology of the vertebral bodies. These were defined by, firstly, examining the inferior border of the vertebral bodies to determine whether they were flat or concave, and subsequently evaluating the shape of C3 and C4: the shape of vertebral bodies transform in a characteristic sequence, developing from trapezoidal to rectangular horizontal, square, and to rectangular vertical. Typically, cervical stages CS1 and CS2 are considered prepubertal; CS3 and CS4, circumpubertal; and CS5 and CS6, postpubertal. 11,12 The individuals in the sample were assigned to one of the six stages of maturation, i.e. CS1, CS2, CS3, CS4, CS5 and CS6, respectively. Subsequently, these six stages of cervical maturation were divided into two groups (Group A and Group B) according to the peak growth, which was considered to be between stages CS3 and CS4. 11,12 The diagnosis of third molar agenesis was performed by using the initial first panoramic radiograph. Subsequently, the diagnosis of agenesis of third molars was confirmed or otherwise by analyzing the second panoramic radiograph obtained after 14 years of age. 14 Correlations between the accuracy of the diagnosis and chronological, dental and or skeletal ages were performed. In this study, the third molar was considered congenitally missing when there was no evidence of radio-transparency associated with the formation of the osseous crypt 14 on panoramic radiography or there was no evidence that the tooth had been extracted. 13,15 Dumas M, Mariano-Pereira P, Bugaighis I, Fernandes-Retto P, Proença L -Criteria for early diagnosis of third molar agenesis: a retrospective radiographic study

ASSESSMENT OF METHOD ERROR
An expert on CVM analysis arranged a training session for the examiner. 13 Subsequently, a calibration test was undertaken.
Thirty-six randomly selected cephalometric radiographs (not included in the present study) were re-analysed after a twoweek interval, by the examiner and the expert. A kappa test revealed almost perfect inter-examiner agreement (k=0.866).
A kappa agreement test was also used to determine the reproducibility of the examiner's assessments, resulting a value of k=0.830. Examiner error was re-determined after a four-week interval for 15% randomly selected cases from the study group for all the analyzed parameters (agenesis of third molars, dental age and skeletal age). The results revealed 100% agreement between the two trials.

STATISTICAL ANALYSIS
Statistical analysis was performed using the software IBM SPSS ® Statistics v. 24 (Armonk, NY, USA). To test the hypotheses initially formulated, the chi-square test of independence was used to evaluate the association between the examined parameters and third molar agenesis. A significance level of 5% was established (p < 0.05).

RESULTS
Out of the 2,960 individuals screened, only 282 (9.5%) fulfilled the inclusion criteria (122 males and 160 females). The number of subjects in each group according to their chronological age Dumas M, Mariano-Pereira P, Bugaighis I, Fernandes-Retto P, Proença L -Criteria for early diagnosis of third molar agenesis: a retrospective radiographic study was as follows (Table 1) and 28 (9.9%). Table 2 illustrates that, for half the cohort, all four second molars were unerupted before the age of 14 years (Group I). Table 2 also shows that the proportion of cases in Group I where the diagnosis of third molar agenesis changed was significantly greater than in the group where the second molars had erupted before fourteen years of age (Group II).
This confirms a significant association between the dental age of second molars and a change in diagnosis (p<0.001). The data show that the number of altered diagnoses decreased with increasing stage of CVM up to the last stage (CS6) at the end of the growth peak, where 100% of the cases maintained the same diagnosis after 14 years of age. To make this outcome less confusing and more easily applied clinically, the data were categorized into two groups: Group A, including pre-peak growth data from individuals who had not reached their peak growth (C1, C2 and C3); and Group B, comprising post-peak growth data (C4, C5 and C6) ( Table 4). In Group A, 22 individuals (14.5%) out of 152 had their diagnosis of third molar agenesis changed after the age of 14 years (Table 3). By contrast, only 6 (4.6%) out of 130 individuals in Group B had their diagnosis of third molar agenesis rejected.
A significant disparity between the two groups concerning diagnostic alterations of third molar agenesis was found (p=0.006).    In the present study, only 28 individuals (9.9% of the sample, with equal numbers of both sexes) showed a change in the diagnosis of third molar agenesis between the first and second radiographs. These results are close to the 6.1% found by Rocha. 18 A chi-squared test did not reveal a significant association between gender and diagnostic change. These findings lead to the conclusion that sex has no influence on the early diagnosis of third molar agenesis, which is in agreement with the findings of Bolaños et al. 14  The outcome of the present study is that it was not possible to make a reliable diagnosis of third molar agenesis in individuals between 11 and 13 years of age (first null hypothesis accepted). However, the dental age, defined by the eruption of the four second molars, was found to be a valid diagnostic criterion of third molars agenesis (second null hypothesis rejected).